Ischemia-reperfusion (I/R) injury by way of heart attacks, strokes and diabetes is one of the major causes of morbidity and mortality in the United States. Production of reactive oxygen species (ROS) after reintroduction of oxygenated blood to ischemic tissue is a major precipitating event causing necrosis and apoptosis in I/R injury. The most reactive and toxic ROS is the hydroxyl radical (OH7) formed by the iron- catalyzed Fenton reaction. The majority of chelatable iron capable of catalyzing the Fenton chemistry is normally contained in lysosomes. Based on preliminary studies, it is probable that iron-dependent radical chemistry during oxidative stress leads to lysosomal membrane disruption and release of chelatable iron into the cytosol. Mitochondria then take up this iron via the electrogenic calcium uniporter to promote intramitochondrial OH7 formation, the mitochondrial permeability transition (MPT) and ultimately cell death. The overall goal of the project to better characterize this process and to develop novel and potentially clinically relevant interventions to block this pathophysiological pathway and prevent lethal cell injury. To achieve this, two specific aims are proposed: 1. Screening of tetracycline-derived compounds to test the hypothesis that cytoprotective compounds are blockers of the mitochondrial calcium uniporter. In preliminary experiments, two tetracycline-derived compounds were shown to be protective against hypoxic and I/R injury. This cytoprotection was associated with inhibition of the mitochondrial electrogenic Ca2+/Fe2+ uniporter. Accordingly, a larger panel of tetracycline-derived compounds will be screened to confirm the association of cytoprotection with uniporter inhibition and to identify a pharmacophore for the cytoprotective compounds. Through use of chelators and confocal microscopy of iron- and calcium-indicating fluorophores, the specific roles of iron and calcium uptake to mitochondrial dysfunction during oxidative stress will be determined. 2. Synthesis and evaluation of lysosomal targeted antioxidants (LTAs) as cytoprotectants against iron- dependant oxidative injury. Antioxidant protection of lysosomes should prevent lysosomal disintegration and iron release and thereby decrease cytosolic iron available for mitochondrial uptake and ROS formation during oxidative stress. As no such LTA currently exists, LTAs will be synthesized and tested for their ability to localize in lysosomes and prevent lysosomal membrane disruption and iron release after acetaminophen- induced oxidative stress. LTAs and cytoprotective tetracycline-derived compounds will also be tested for their ability to protect against acetaminophen toxicity and I/R injury. Overall, these studies will lead to a better understanding of the interrelationships of lysosomal and mitochondrial dysfunction in oxidative, I/R and hepatotoxic stress and lead to new therapeutic interventions to minimize cell and tissue damage. PUBLIC HEALTH RELEVANCE: Ischemia/reperfusion injury occurring in heart attack, stroke and complications of diabetes is one of the leading causes of suffering and death in the United States. Iron appears to predispose these diseases. My project proposes to better understand the role of iron mobilization from lysosomes to mitochondria in ischemia/reperfusion injury and to develop novel therapies to mitigate or eliminate damage from this condition.